Summary: Although the majority of early stage estrogen receptor (ER)-positive breast cancers are cured through multimodality care, metastatic ER-positive breast cancer remains a lethal disease. Insights into this discrepancy have come through comparative genomic analyses of primary and metastatic tumors. We and others have identified several mutations affecting specific genes that are more prevalent in metastatic cancers than in their primary counterparts, including ESR1, ERBB2 and NF1. These mutations result in resistance to front-line endocrine treatments that are the mainstay systemic therapy in ER-positive breast cancer. Even more striking than such individual mutations, however, has been the finding that certain `mutational signatures' are enriched in metastatic disease as compared to primary breast cancers. These mutational signatures represent the DNA damage and repair processes that shape the cancer genome and can give rise to such mutations and the transformed phenotypes they convey. A glaring and consistent finding from multiple large-scale sequencing studies has been that the APOBEC mutational signature is both enriched and highly prevalent in ER-positive metastatic disease, comprising the dominant mutational signature for these drug-resistant and ultimately lethal cancers. Our preliminary data confirm that APOBEC activation can promote the development of endocrine resistance in ER-positive cancer models and is associated with characteristic APOBEC-mutational changes in many drug resistance alleles. Together, these results point to the APOBEC mutational process as a key driver in the development and pathogenesis of ER-positive metastatic breast cancer and endocrine therapy resistance. In this highly collaborative and innovative project, we propose three specific aims to advance the APOBEC mutational process as a biomarker and therapeutic target in breast cancer. (1) We will develop and utilize robust bioinformatic methods to detect the presence and the timing of onset of the APOBEC mutational signature from clinical NGS datasets of both tumor and cell free DNA (cfDNA). We will further ascertain if a promising IHC assay for the A3B enzyme can identify those ER-positive cancers likely to subsequently develop an APOBEC mutational signature. (2) We will determine the mechanisms and kinetics of APOBEC's contribution to endocrine resistance. We will use isogenic cell line models and patient derived xenografts to dissect the types of resistance patterns that are caused by APOBEC as well their timing and whether the endocrine therapy itself contributes to the induction of APOBEC activity. (3) We will assess both candidate and unbiased synthetic lethal approaches to targeting tumors in which APOBEC activity is induced and determine their capabilities in killing APOBEC- positive cells and preventing the development of endocrine resistance. We anticipate that our findings will uniquely position our team to launch clinical trials testing specific approaches to diagnose APOBEC-positive tumors, to prevent the development of resistance to endocrine therapies, and to target the largest subset of ER- positive endocrine-resistant metastatic breast cancers.